The enzyme dopamine-beta-hydroxylase (DBH) occupies a central role in the pathway in which tyrosine is converted to the neurotransmitter norepinephrine. Since approximately 90 percent of this enzyme is bound to the membrane of the adrenergic storage granule, it is an excellent marker for this membrane. Determination of DBH activity, after sucrose density gradient centrifugation, makes it possible to locate and identify specific populations of storage granules. Experiments will be carried out to examine questions with regard to the functional role of the different populations of vesicle. In other experiments the possibility that vesicles are reused will be examined as will the possibility that the small granular vesicles are derived from the large or dense granular vesicles. The use of organ culture techniques makes it possible to assess several of the potential regulatory mechanisms which may be acting to alter the level of either DBH or of tyrosine hydroxylase (TH). Using this system we will examine the effect of cyclic nucleotides, nerve growth factor, and electrical stimulation. Organ culture will also be used to study the interaction between sympathetic nerves and their target organs and to study the factors which regulate the development of sympathetic ganglia. There are several factors which may potentially limit the expression of DBH activity in vivo. We will investigate the possibility that access of dopamine to DBH, which is sequestered in adrenergic storage granules, is a limiting factor in norepinephrine biosynthesis. In another series of experiments we will investigate the endogenous inhibitors of DBH which are present in a variety of adrenergically innervated organs. These inhibitors will be purified from several organs and an attempt will be made to assess their role in the regulation of DBH activity and thus of norepinephrine biosynthesis.